PROJECT SUMMARY The number of Americans over age 65 is projected to increase from approximately 39 million in 2010 to an estimated 71 million in 2030 (2010 census). While only 13% of US population is 65 and older, they consume over 36% of personal health expenditures. Cardiovascular disease (CVD) is a major driver of morbidity, mortality, and health care costs in the elderly population. The cardiovascular system is prone to age-dependent deterioration even in the absence of traditional risk factors, and although these changes have been extensively characterized, the underlying mechanisms are incompletely understood. The functional and structural changes that occur during biological aging, including endothelial dysfunction, reduced vessel elasticity, and chronic vascular inflammation, ultimately result in decreased vascular compliance, systemic hypertension, arterial thrombosis and ischemic events. The previous iterations of this grant supported an investigative program that helped to define the molecular regulation of plasminogen activator inhibitor-1 (PAI-1) and its role in the development of vascular pathology. Based on work from this laboratory, PAI-1 is now recognized to have broad biologic relevance beyond its role in thrombosis. There is compelling evidence that senescent cells accumulate in tissues and contribute to the aging. In addition to contributing to the molecular fingerprint of senescence, PAI-1 is necessary and sufficient for the induction of replicative senescence in vitro. We have significantly advanced our understanding the role of PAI-1 as a potential driver of cellular and vascular senescence, as well as biological aging in murine models. Importantly, our initial characterization of lifespan, cardiovascular function, and markers of senescence in a unique kindred of Amish individuals harboring a loss of function mutation in the gene encoding PAI-1 (SERPINE1) reveals improved health span and longevity in humans with heterozygous PAI-1 deficiency compared to unaffected age-matched kindred. Based on these collective observations we hypothesize that PAI-1 promotes vascular senescence and aging-related cardiovascular morbidity. We propose a multifaceted phylogenetically diverse program using Drosophila, mice and humans, to delineate the involvement of PAI-1 in age-related vascular remodeling across species. We anticipate that these studies will not only establish the role of PAI-1 in senescence, but will also advance our knowledge of the mechanisms that drive physiological aging. Furthermore, understanding the molecular mechanism of PAI-1's role in senescence and aging may provide new insights into the prevention and treatment of aging-related dysfunction and frailty.